Agar is a complex mixture of polygalactans obtained from red algae, having a basic disaccharide repeat unit: 3-O-(3,6-anhydro-L-galactopyranosyl -!

-"-galactopyranose #hich can be substituted #ith methyl ($-6 of "-unit, $-% of L-unit or #ith sulfate ($-6 og Lunit & 'he neutral form of these polysaccharides is called agarose: $ommercial grade agar and agarose are mainly obtained from Gelidium and Gracilaria species& 'he initial approach of our laboratory #as the development of thalli cultivation, agar extraction and analytical procedures oriented to the production and the study of agarose& (ince then several other forms have been observed for these polymers, as earlier studies predicted () , especially #hen their distribution #ithin main axis and lateral branches #ere ta*en into consideration& +n order to verify the hypothesis that all these polymers reflect different steps of agar metabolism (% , it #as necessary to revie# the methodology of structural investigations and studies of their heterogeneity& +n this #or*, special attention #as given to the substituents of "-galactose units since this residue is not only the site of O-methylation at the $-6 (6-O-methyl-L-galactose, 6-O--e-gal (3 & .e have compared the #ater (/ and ethanol0#ater extractions and hace developed a ne# extraction procedure (1igure ) #hich fractionates the polymers according to their degree of substitution (sulfation, methylation & -ain axis (-A and lateral branches (L! from Gracilaria verrucosa #ere cultivated separately under promoting and non-promoting gro#th conditions& 'he monosaccharide content of the polymers #as obtained by 2L$ analysis of their alditol acetates (6 & 'he sulfate content #as measured by and 34L$ method (5 & 3,6-anhydrogalactose and total carbohydrate contents #ere estimated by colorimetric assays (6,7 & 'he evolution of the polymers #as monitored by comparison #ith the structural data of the corresponding fractions obtained from native plants (table + & 'he degree of substitution of the "-unit #as computed from the 6-O-methyl-gal and /-O-methyl contents& At the end of the cultivation cycle (% months , the only significant variation in the composition of -A, #as an increase of /-O-methyl-gal content gro#th-promoting conditions as earlier described for 2racilaria ti*vahiae ()8 , #hile L! represented a remar*able increase in 6-O-methyl-gal& +t is interesting to note that the degree of substitution ("( remained the same in both cases& 9nder non-promoting conditions no significant variation #as observed, except a slight increase of 6-O-methyl-gal in L!& !etter recovery #as obtained #ith the ethanol0#ater extraction, #here :A #as the ma;or fraction& 1or the #ater extraction, .! #as the main fraction (figure % & 'he yield of hot soluble polysaccharides (:A, :!, .A and .! #as greater from L! than from -A& 'he 6-O--e-gal0 /-O--e-gal ratio decreased during the extraction step, the lo#est value being obtained for algal residues in all cases& An inverse relationship #as observed

bet#een this ratio and the sulfate content (data not sho#n & As the /-O--e-gal content is the highest for the algal residues, this sugar #as demonstrated to belong to neutral type polysaccharides closely bound to the cell #all and thus having lo# extractability& Our results have further allo# us to demonstrated the implication of /-O-methyl-galactose in the agar metabolism and a continuous exchange bet#een O-methylation and /-Omethyl-galactose lin*age to the "-galactose units during maturation of thalli ())-)7 &

References

)& +<umi, =& )75%& $hemical heterogeneity of the agar from 2racilaria verrucosa& >& !iochem& >ap&, 5% () : )3?-)/8& %& $hristiaen ", ' (tadler, -A Ondar<a @ -$ Aerdus& )765& (tructures and functions of the polysaccharide from the cell #all of Gracilaria verrucosa (Bhodophyceae, 2igartinales & 3ydrobiologia )?)0)?%: )37-)/6& 3& =aramanos C, -A Ondar<a, 1 !ellanger, " $hristiaen @ ( -oreau& )767& 'he lin*age of /-O-methyl Lgalactopyranose in the agar polymers from Gracilaria verrucosa. $arbohydrate Besearch )65: 73-)8)& /& $hristiaen "& '&, =aramanos, C& , (tadler, '& and 3& -orvan& ()765 & 1ractions polysaccharidiDues extradites de la paroi de 2racilaria verrucosa& (ci& de l Eau, 6, )%)-)3?& ?& Lahaye -, $ Bochas @ . Caphe& )766& A ne# procedure for determining the heterogeneity of agar polymers in the cell #alls of Gracilaria sp& (2racilariaceae, Bhodophyta & $an& >&!ot& 6/: ?57-?6?& 6& (lone*er >3& )75%& 2as-liDuid chromatography of alditol acetates& :n: .histler BL @ >F !emiller& -ethods in $arbohydrate $hemistry 6: %8-%/& Academic 4ress, Fe# Cor*& 5& -aas, 1&-&, 3offmann, +&, Aan 3armelen, -&(& and L&(& de =o*& )766& Befractometric determination of sulphate and other anions in plants separated by 34L$& 4lant @ (oil, 7): )%7-)3%& 6& "ubois, -&, 2illes, 1&A&, 3amilton, >&=&, Bebers, 4&A& and (mith, 1& ()7?6 & $olorimetric method for the determination of sugars and related substances& Anal& !iochem&, %6, 3?8-3?6& 7& Caphe, .& )768& $olorimetric determination of 3,6-anhydroga- lactose and gGlactose in marine algal poloysaccharides& Anal& $hem& 3%: )3%5-)338& )8& $raigie, >&(&, and H&$& .en&)76/ :ffects of temperature and tissue age on gel strength and composition of agar from 2racilaria ti*vahiae (Bhodophyceae & $an& >& !ot& 6%:66?I)658& ))& =imon '& !ird& )778& :vidence of /-O-methyl-J-L-galactose in the agar of Gracilaria verrucosa strain 2-)6& )7, >ournal of Applied 4hycology Aolume %, Fumber /, 363-36/&

12. >&(& $raigie and A& >urgens& )767& (tructure of agars from Gracilaria tikvahiae rhodophyta: Location of 4-O-methyl-L-galactose and sulphate& $arbohydrate 4olymers& Aolume )), +ssue /, 4ages %6?-%56& )3& O& Aaliente, L& :& 1ernande<, B& -& 4ere<, 2& -arDuina and 3& Aele<& )77%& Agar 4olysaccharides from the Bed (ea#eeds 2racilaria domingensis (onder ex =Kt<ing and 2racilaria mammillaris (-ontagne 3o#e& !otanica -arina& Aolume 3?, +ssue %, 4ages 55I6)& )/& >&$& -ollet, -&$& Aerdus, B& =ling and 3& -orvan&& )77?& +mproved protoplast yield and cell #all regeneration in Gracilaria verrucosa (3uds& 4apenfuss (2racilariales, Bhodophyta & >ournal of :xperimental !otany& Aolume/6, +ssue%, 4p& %37-%/5& )?& -& Lahaye,>& 1& Bevol,$& Bochas,>& -cLachlan and.& Caphe& )766 & 'he $hemical (tructure of 2racilaria crassissima (4& et 3& $rouan in (chramm et -a<E 4& et 3& $rouan in (chramm et -a<E and 2& ti*vahiae -cLachlan (2igartinales, Bhodophyta $ell-.all 4olysaccharides& !otanica -arina& Aolume 3), +ssue 6, 4ages /7)I?8%& )6& -& Lahaye and .& Caphe& )767 & 'he $hemical (tructure of Agar from 2racilaria compressa ($& Agardh 2reville, 2& cervicornis ('urner >& Agardh, 2& damaecornis >& Agardh and 2& domingensis (onder ex =Kt<ing (2igartinales, Bhodophyta !otanica -arina& Aolume 3%, +ssue /, 4ages 367I356& )5& Bonald "& Aillanueva, >umelita !& Bomero, Anita Linda B& Bagasa and -arco Femesio :& -ontaLo& %8)8& Agar from the red sea#eed, Laurencia flexilis ($eramiales, Bhodophyta from northern 4hilippines& 4hycological Besearch& Aolume ?6, +ssue %, pages )?)I)?6& )6& Femesio :& -ontaLo, Bonald "& Aillanueva and >umelita !& Bomero& )777& $hemical characteristics and gelling properties of agar from t#o 4hilippine 2racilaria spp& (2racilariales, Bhodophyta >ournal of Applied 4hycology Aolume )), Fumber ), %5-3/& 19. C& 1reile-4elegrMn and "& Bobledo& )775& :ffects of (eason on the Agar $ontent and $hemical $haracteristics of 2racilaria cornea from CucatNn, -Exico& !otanica -arina& Aolume /8, +ssue )-6, 4ages %6?I%78&